During the development of muscle, contractile proteins are first detected in the multinucleated cells formed by the fusion of single myoblasts. The nuclei of these differentiating muscle cells no longer synthesize DNA. In culture, the time of initiation of fusion is controlled by diffusion-mediated processing of the medium which also retards cell proliferation. Although the myoblasts continue to synthesize DNA, the distribution of generation times is more variable due to protraction of G1. We have shown that cells which fuse do spend more time in G1 than cells still in the cycle, but that the distributions of these values overlap. This study will be extended to determine whether the shorter distribution of G1 in cycling cells is due to loss by fusion of the absent cells with longer G1s. To this end the distribution of G1 will be measured in low Ca ions medium (which prevents fusion). During this grant year we have been able to demonstrate, by continuous perfusion, that cell fusion can occur even though cell products are prevented from accumulating in the medium. This technique will be applied next to determine critically whether fusion, in this circumstance, is dependent on growth limitation. Perfusion techniques will also be used evaluate whether withdrawal of myoblasts from the cell cycle in low Ca plus medium is due to the absence of sufficient Ca plus per se or to depletion of growth factors. Studies of cell synchrony will be continued to determine whether or not cell type specific biosynthesis occurs during the G1 phase of every cycle and of itself does not preclude re-entry into the cycle. Our studies of the regeneration of myoblasts from adult fibers will be extended to adult mammals and amphibians with the ultimate goal of determining whether myogenic cells can be derived from these species and whether such cell population have different properties from embryonic myoblasts suggestive of maturational changes. Bibliographic references: Konigsberg, U.R., B. H. Lipton and I. R. Konigsberg (1975). "The regenerative response fo single mature muscle fibers isolated in vitro." Develop. Biol. 45: 260-275.